Analysis of the long control region of bovine papillomavirus type 1 associated with sarcoids in equine hosts indicates multiple cross-species transmission events and phylogeographical structure.

Papillomaviruses are a family of slowly evolving DNA viruses and their evolution is commonly linked to that of their host species. However, whilst bovine papillomavirus-1 (BPV-1) primarily causes warts in its natural host, the cow, it can also cause locally aggressive and invasive skin tumours in equids, known as sarcoids, and thus provides a rare contemporary example of cross-species transmission of a papillomavirus. Here, we describe the first phylogenetic analysis of BPV-1 in equine sarcoids to our knowledge, allowing us to explore the evolutionary history of BPV-1 and investigate its cross-species association with equids. A phylogenetic analysis of the BPV-1 transcriptional promoter region (the long control region or LCR) was conducted on 15 bovine and 116 equine samples from four continents. Incorporating previous estimates for evolutionary rates in papillomavirus implied that the genetic diversity in the LCR variants was ancient and predated domestication of both equids and cattle. The phylogeny demonstrated geographical segregation into an ancestral group (African, South American and Australian samples), and a more recently derived, largely European clade. Whilst our data are consistent with BPV-1 originating in cattle, we found evidence of multiple, probably relatively recent, cross-species transmission events into horses. We also demonstrated the high prevalence of one particular sequence variant (variant 20), and suggest this may indicate that this variant shows a fitness advantage in equids. Although strong host specificity remains the norm in papillomaviruses, our results demonstrate that exceptions to this rule exist and can become epidemiologically relevant.

Equine sarcoids: Bovine Papillomavirus type 1 transformed fibroblasts are sensitive to cisplatin and UVB induced apoptosis and show aberrant expression of p53.

Bovine papillomavirus type 1 infects not only cattle but also equids and is a causative factor in the pathogenesis of commonly occurring equine sarcoid tumours. Whilst treatment of sarcoids is notoriously difficult, cisplatin has been shown to be one of the most effective treatment strategies for sarcoids. In this study we show that in equine fibroblasts, BPV-1 sensitises cells to cisplatin-induced and UVB-induced apoptosis, a known cofactor for papillomavirus associated disease, however BPV-1 transformed fibroblasts show increased clonogenic survival, which may potentially limit the therapeutic effects of repeated cisplatin treatment. Furthermore we show that BPV-1 increases p53 expression in sarcoid cell lines and p53 expression can be either nuclear or cytoplasmic. The mechanism and clinical significance of increase/abnormal p53 expression remains to be established.

A multimeric L2 vaccine for prevention of animal papillomavirus infections.

It is unclear what level of neutralizing antibody is sufficient to protect cattle from experimental bovine papillomavirus type 4 (BPV4) challenge. Markedly lower, and often undetected, serum neutralizing antibody titers were associated with protection in cattle vaccinated with BPV4 L2 as compared to L1 VLP. We hypothesized that vaccination with concatemers of the N-terminal protective epitopes of L2 derived from multiple animal papillomavirus types would enhance the breadth and strength of immunity. Therefore we generated a multimeric L2 antigen derived from three bovine and three canine papillomavirus types with divergent phenotypes and purified it from bacteria. Mice vaccinated three times with this six type L2 vaccine formulated in alum or RIBI adjuvant generated robust serum neutralizing antibody titers against BPV1, BPV4 and canine oral papillomavirus (COPV). Furthermore, vaccination with this six type L2 vaccine formulated in adjuvant, like BPV1 L1 VLP, protected the mice from experimental challenge with BPV1 pseudovirus.

Upregulation of equine matrix metalloproteinase 1 by bovine papillomavirus type 1 is through the transcription factor activator protein-1.

Equine sarcoids represent the most common skin tumours in equids worldwide, characterized by extensive invasion and infiltration of lymphatics, rare regression and high recurrence after surgical intervention. Bovine papillomavirus type 1 (BPV-1) activity is necessary for the transformation phenotype of equine fibroblasts. Among the many changes induced by BPV-1, matrix metalloproteinase 1 (MMP-1) upregulation contributes to the invasiveness of equine fibroblasts. However, it is not yet known how BPV-1 proteins regulate equine MMP-1 expression. To elucidate this mechanism, the equine MMP-1 promoter was cloned and analysed. A putative activator protein-1 (AP-1)-binding site was demonstrated to be crucial for upregulated MMP-1 promoter activity by BPV-1. BPV-1 E6 and E7 proteins increased MMP-1 promoter activity, and inhibition of BPV-1 gene expression by small interfering RNA significantly reduced the promoter activity. c-Jun and Fra-1, two components of the AP-1 transcription factor complex, were overexpressed and activated by BPV-1 in equine fibroblasts. Finally, BPV-1 E5, E6 and E7 proteins increased MMP-1 mRNA and protein expression. In conclusion, the expression of MMP-1 can be enhanced by BPV-1 oncoproteins E6 and E7 through the AP-1 transcription factor and by E5 via an indirect mechanism. These findings shed light on the mechanism of BPV-1-mediated equine fibroblast infiltration and indicate that both BPV-1 oncoproteins and AP-1 could be potential targets for equine sarcoid therapy.

p38 mitogen-activated protein kinase is crucial for bovine papillomavirus type-1 transformation of equine fibroblasts.

Equine sarcoids represent the most common skin tumours in equids worldwide, characterized by extensive invasion and infiltration of lymphatics, rare regression and high recurrence after surgical intervention. Bovine papillomavirus type-1 (BPV-1) and less commonly BPV-2 are the causative agents of the diseases. It has been demonstrated that BPV-1 viral gene expression is necessary for maintaining the transformation phenotype. However, the underlying mechanism for BPV-1 transformation remains largely unknown, and the cellular factors involved in transformation are not fully understood. Previously mitogen-activated protein kinase (MAPK) signalling pathway has been shown to be important for cellular transformation. This study investigated the role of p38 MAPK (p38) in the transformation of equine fibroblasts by BPV-1. Elevated expression of phosphorylated p38 was observed in BPV-1 expressing fibroblasts due to the expression of BPV-1 E5 and E6. The phosphorylation of the MK2 kinase, a substrate of p38, was also enhanced. Inhibition of p38 activity by its selective inhibitor SB203580 changed cell morphology, reduced the proliferation of sarcoid fibroblasts and inhibited cellular invasiveness, indicating the indispensable role of p38 in BPV-1 transformation of equine fibroblasts. These findings provide new insights into the pathogenesis of equine sarcoids and suggest that p38 could be a potential target for equine sarcoid therapy.

Different contribution of bovine papillomavirus type 1 oncoproteins to the transformation of equine fibroblasts.

Equine sarcoids represent the most common skin tumours in equids worldwide, characterized by localized invasion, rare regression and high recurrence following surgical intervention. Bovine papillomavirus type 1 (BPV-1) and less commonly BPV-2 are now widely recognized as the causative agents of the disease. Fibroblasts isolated from sarcoids are highly invasive. Invasion is associated with a high level of viral gene expression and matrix metalloproteinase upregulation. However, it remains unclear to what extent BPV-1 proteins are involved in the transformation of equine cells. To address this question, the individual viral genes E5, E6 and E7 were overexpressed in normal equine fibroblasts (EqPalF cells) and in the immortal but not fully transformed sarcoid-derived EqS02a cell line. The proliferation and invasiveness of these cell lines were assessed. E5 and E6 were found to be responsible for the enhanced cell proliferation and induction of increased invasion in EqS02a cells, whilst E7 appeared to enhance cell anchorage independence. Knockdown of BPV-1 oncogene expression by small interfering RNA reversed the transformed phenotype of sarcoid fibroblasts. Together, these observations strongly suggest that BPV-1 proteins play indispensable roles in the transformation of equine fibroblasts. These data also suggest that BPV-1 proteins are potential drug targets for equine sarcoid therapy.

1st International Workshop on Papillomavirus E5 Oncogene-a report.

The 1st International Workshop on Papillomavirus E5 Oncogene was held in Capri, Italy, 27-28 May 2010. Here we present a brief report of the various lectures which addressed the multiple facets of the E5 protein.

All 4 di-leucine motifs in the first hydrophobic domain of the E5 oncoprotein of human papillomavirus type 16 are essential for surface MHC class I downregulation activity and E5 endomembrane localization.

The E5 oncoprotein of human papillomavirus type 16 downregulates surface MHC Class I and interacts with the heavy chain of the MHC complex via the first hydrophobic domain, believed to form the first helical transmembrane region (TM1) of E5. TM1 contains 4 equally spaced di-leucine (LL1-LL4) motifs. Di-leucine motifs have been implicated in protein-protein interactions and as localization signals. To see if any of the 4 di-leucine motifs of TM1 are involved in MHC downregulation by E5, we mutated each LL pair into valine pairs (VV1-VV4), as mutation of leucine to valine is not expected to cause major structural alterations in E5. We found that all 4 mutations disrupted the intracellular location of E5 and abrogated its MHC I downregulating activity; however VV2 and VV4 mutants were still able to interact physically with the MHC I heavy chain (HC) in vitro, while VV1 and VV3 mutants had lost this activity. We conclude that LL1 and LL3 are necessary for the interaction with HC, but LL2 and LL4 are not. However all 4 LL motifs are responsible for the proper localization of E5 in the Golgi/ER, and the displacement of E5 from this location contributes to the abrogation of MHC I downregulation. LL1 and LL3 motifs are expected to be on one face of the TM1 helix and LL2 and LL4 on the opposite face. We propose that E5 interacts with HC via LL1 and LL3 and that all 4 di-leucine motifs act as a targeting signal.

Equine sarcoid fibroblasts over-express matrix metalloproteinases and are invasive.

Papillomaviruses are DNA viruses that cause tumours of the skin in humans and animals. The natural host of bovine papillomavirus is cattle, but also equids, resulting in tumours termed sarcoids. Matrix metalloproteinase 1 (MMP-1) expression is up-regulated in sarcoid fibroblasts and tumours. We extended our observation to other MMPs and determined whether MMPs induced invasion of sarcoid fibroblasts. Collagenase (MMP-1) and Gelatinase (MMP-2, MMP-9) were over-expressed in sarcoid fibroblasts and tumours. The fibroblasts were invasive in a 3D/matrigel invasion assay system. Inhibition of MMP by GM6001 significantly reduced invasion. E2 siRNA treatment of sarcoid fibroblasts decreased the expression of the viral genes and of MMP-2 and -9, leading to a dramatic reduction of invasion. This demonstrates that BPV-1 induces over-expression of MMPs contributing to invasiveness of sarcoid fibroblasts. Inhibition of E2 by siRNA leads to abrogation of invasion suggesting that E2 is a good target for sarcoid treatment.

Papillomavirus prophylactic vaccines: established successes, new approaches.

Vaccines against the human papillomaviruses (HPVs) most frequently associated with cancer of the cervix are now available. These prophylactic vaccines, based on virus-like particles (VLPs), are extremely effective, providing protection from infection in almost 100% of cases. However, the vaccines present some limitations: they are effective primarily against the HPV type present in the vaccine, are expensive to produce, and need a cold chain. Vaccines based on the minor capsid protein L2 have been very successful in animal models and have been shown to provide a good level of protection against different papillomavirus types. The potential of L2-based vaccines to protect against many types of HPVs is discussed.

Effects of human papillomavirus type 16 E5 deletion mutants on epithelial morphology: functional characterization of each transmembrane domain.

Human papillomavirus type 16 (HPV-16) is the cause of cervical cancer. The HPV genome encodes three transforming proteins, E5, E6 and E7. E6 and E7 are the main transforming proteins of HPV, while the role of E5 is still poorly understood. Using three dimensional organotypic raft cultures we show that HaCaT human keratinocytes expressing HPV-16 E5 form a very perturbed epithelium, with simultaneous hyperkeratinization of some cells and defective differentiation of other cells. The basal layer is disturbed and many cells invade the collagen matrix. Many cells among the differentiated layers show characteristics of basal cells: progression through the cell cycle, expression of cytokeratin 14, lack of cytokeratin 1 and production of matrix metalloproteases (MMP). Using deletion mutants which encompass the three hydrophobic domains of E5, we have assigned the ability to promote invasion of the matrix to the first hydrophobic domain, and the capacity to induce MMP9 to the C-terminal four amino acids. We also show that invasion and production of MMP9 can be dissociated, as mutants that are still capable of invasion do not produce MMP9 and vice versa.

Bovine papillomavirus type 1 oncoprotein E5 inhibits equine MHC class I and interacts with equine MHC I heavy chain.

Bovine papillomavirus type 1 is one of the aetiological agents of equine sarcoids. The viral major oncoprotein E5 is expressed in virtually all sarcoids, sarcoid cell lines and in vitro-transformed equine fibroblasts. To ascertain whether E5 behaves in equine cells as it does in bovine cells, we introduced the E5 open reading frame into fetal equine fibroblasts (EqPalF). As observed in primary bovine fibroblasts (BoPalF), E5 by itself could not immortalize EqPalF and an immortalizing gene, such as human telomerase (hTERT/hT), was required for the cells to survive selection. The EqPalF-hT-1E5 cells were morphologically transformed, elongated with many pseudopodia and capable of forming foci. Equine major histocompatibility complex class I (MHC I) was inhibited in these cells at least at two levels: transcription of MHC I heavy chain was inhibited and the MHC I complex was retained in the Golgi apparatus and prevented from reaching the cell surface. We conclude that, as in bovine cells and tumours, E5 is a player in the transformation of equine cells and the induction of sarcoids, and a potential major cause of MHC I downregulation and hence poor immune clearance of tumour cells.

Small interfering RNA targeting bovine papillomavirus type 1 E2 induces apoptosis in equine sarcoid transformed fibroblasts.

Equine sarcoids are skin tumours of horses caused by infection with BPV-1 or 2. Maintenance and replication of the viral genome depend upon the viral proteins E1 and E2. We examined the effects of an E2 specific siRNA on E2 and E1 viral gene expression, viral load and cell growth in BPV-1 transformed sarcoid-derived cells. Transfection with E2-siRNA caused a reduction in E2 and E1 mRNA expression as well as viral load, growth inhibition and decreased anchorage-independent growth. siRNA treated cells showed significantly higher apoptosis rates than control cells. Thus sequence specific targeting of E2 provides a powerful strategy to eliminate BPV-1 genomes and induce cell death in BPV-1 transformed cells.

The detection of Bovine Papillomavirus type 1 DNA in flies.

BPVs are double stranded DNA viruses that can infect several species other than the natural host, cattle, including equids. In equids, BPV-1, and, less commonly BPV-2, infection gives rise to fibroblastic tumours of the skin. Whilst a causal relationship between BPV-1/2 and equine sarcoids is now well established, how the disease is transmitted is not known. In this study we show BPV-1 DNA can be detected in flies trapped in the proximity of sarcoid-affected animals. Sequence analysis of the BPV-1 LCR from flies indicates that flies harbour BPV-1 LCR sequence variants II and IV which are commonly detected in equine sarcoids. These data suggest that flies may be able to transmit BPV-1 between equids.

Bovine papillomaviruses: their role in the aetiology of cutaneous tumours of bovids and equids.

Bovine papillomavirus (BPV) is perhaps the most extensively studied animal papillomavirus. In cattle BPVs induce benign tumours of cutaneous or mucosal epithelia, called papillomas or warts. Cattle papillomas are benign tumours and generally regress without eliciting any serious clinical problems in the host, but occasionally persist and provide the focus for malignant transformation to squamous cell carcinoma, as in the case of cancer of the urinary bladder and cancer of the upper alimentary canal. BPV is the only papillomavirus that jumps species: the virus also infects equids, and gives rise to fibroblastic tumours called sarcoids. Sarcoids very rarely regress, more often they persist and can be locally aggressive. These tumours are the most common dermatological tumour of equids worldwide. The purpose of this review is to discuss the biology of BPV, the biology of bovine tumours and equine sarcoids, and present the current understanding of BPV in tumour pathogenesis in its natural host, cattle, and in its heterologous host, equids. Finally, the use of anti-BPV vaccines as a therapy for equine sarcoids will be discussed. Only limited information on the clinical or pathological aspects of either bovine or equine tumours will be provided as this subject has been extensively addressed previously.

The E6E7 oncoproteins of cutaneous human papillomavirus type 38 interfere with the interferon pathway.

Non-melanoma skin cancer is the most frequent malignancy in Caucasian populations. Evidence suggests the involvement of cutaneous Human Papillomavirus (HPV) of the genus beta (beta) in this disease. The ability of E6 and E7 of mucosal HPV to promote cellular transformation and inhibit immune response-related pathways plays a key role in cervical carcinogenesis. beta HPV-38 E6 and E7 display transforming activities in in vitro and in vivo models, but their impact on immune surveillance is unknown. Here we show that HPV-38 E6 and E7 affect the IFN-induced up-regulation of MHC class I. Expression of the two viral proteins in HaCaT keratinocytes led to a decrease of MHC I levels. This down-regulation is associated with a reduction of expression of MHC I heavy chain, of the peptide chaperone TAP and of the STAT-1 downstream effector IRF-1. The down-regulation of these proteins is ultimately due to the inhibition of STAT-1 expression. Analysis of cells expressing either HPV-38 E6 or E7 suggests that these effects are primarily the result of E6 expression, although a contribution by E7 cannot be excluded. We conclude that HPV-38 encodes oncoproteins that potentially contribute to the evasion of host immune surveillance.

Comparative transforming potential of different human papillomaviruses associated with non-melanoma skin cancer.

It is well established that high-risk human papillomaviruses (HPVs) that infect mucosal epithelia are the causative agents of cervical cancer. In contrast, the association of cutaneo-tropic HPV types with the development of non-melanoma skin cancer (NMSC) is less well defined. In this study, we have analysed the in vitro transforming potential of various cutaneous HPV types. Using oncogene cooperation assays with activated ras, we have shown that diverse cutaneous types, including 12, 14, 15, 24, 36 and 49, have significant transforming potential. Interestingly, most of this activity appears to be encoded by the E6 gene product. In contrast, the common HPV-10 exhibits no significant transforming potential in these assays. This difference may be a reflection of different patterns of cellular localization, with transforming E6s being nuclear and non-transforming being cytoplasmic. These results provide molecular support for a role of these viruses in the development of certain human malignancies.

Detection of bovine papillomavirus type 1 genomes and viral gene expression in equine inflammatory skin conditions.

Papillomaviruses are normally strictly species-specific and even under experimental conditions do not usually infect any other host than the natural host. The only documented reports of natural papillomavirus cross-species infection are of BPV-1/BPV-2, which can infect horses and induce equine sarcoids. BPV DNA has not been detected in non-sarcoid equine tumours or equine papillomas, but its presence has been reported in some cases of equine dermatitis. In the present study, we show that equine inflammatory skin conditions harbour episomal circular double stranded BPV-1 genomes, with copy numbers ranging from 0.2 to 155 copies/cell. BPV-1 E1, E2 and E5 genes were expressed in these inflammatory skin lesions, indicating active infection. We conclude that some cases of equine dermatitis are associated with the presence of circular, episomally maintained BPV-1 genomes that express viral transcripts.

Bovine papillomavirus infection in equine sarcoids and in bovine bladder cancers.

Bovine papillomavirus (BPV) type 2 is involved in carcinogenesis of the urinary bladder in cattle, while BPV-1 is commonly associated with equine sarcoid tumours. In both cases the early viral proteins are expressed, but virion is not produced. Given the similarities in BPV biology between the tumours in cattle and horses, bovine bladder cancers and equine sarcoids were compared with respect to physical status, load of viral DNA and variability of the E5 open reading frame (ORF). Rolling circle amplification demonstrated that BPV-1 and BPV-2 genomes exist as double stranded, episomal, circular forms in the two tumours. Realtime quantitative PCR revealed that equine sarcoids contained higher viral DNA loads compared to bovine bladder cancers. The BPV-1 E5 ORF showed sequence variation but BPV-2 ORF did not. The presence of BPV-1 E5 variations or their absence in the BPV-2 E5 ORF does not appear to have an effect on viral DNA load in either tumour type.